Hypertonic Saline Is Effective in the Prevention and Treatment of Mucus Obstruction, but Not Airway Inflammation, in Mice with Chronic Obstructive Lung Disease

Recent evidence suggests that inadequate hydration of airway surfaces is a common mechanism in the pathogenesis of airway mucus obstruction. Inhaled hypertonic saline (HS) induces osmotic water flux, improving hydration of airway surfaces. However, trials in patients with obstructive lung diseases are limited. The aim of this study was to investigate effects of HS on mucus obstruction and airway inflammation in the prevention and treatment of obstructive lung disease in vivo. We, therefore, used the β-epithelial Na+ channel (βENaC)–overexpressing mouse as a model of chronic obstructive lung disease and determined effects of preventive and late therapy with 3% HS and 7% HS on pulmonary mortality, airway mucus obstruction, and inflammation. We found that preventive treatment with 3% HS and 7% HS improved growth, reduced mortality, and reduced mucus obstruction in neonatal βENaC-overexpressing mice. In adult βENaC-overexpressing mice with chronic lung disease, mucus obstruction was significantly reduced by 7% HS, but not by 3% HS. Treatment with HS triggered airway inflammation with elevated keratinocyte chemoattractant levels and neutrophils in airways from wild-type mice, but reduced keratinocyte chemoattractant in chronic neutrophilic inflammation in adult βENaC-overexpressing mice. Our data demonstrate that airway surface rehydration with HS provides an effective preventive and late therapy of mucus obstruction with no consistent effects on inflammation in chronic lung disease. These results suggest that, through mucokinetic effects, HS may be beneficial for patients with a spectrum of obstructive lung diseases, and that additional strategies are required for effective treatment of associated airway inflammation

Effects of lumacaftor—ivacaftor therapy on cystic fibrosis transmembrane conductance regulator function in F508del homozygous patients with cystic fibrosis aged 2–11 years

Rationale: Lumacaftor/ivacaftor was approved for the treatment of patients with cystic fibrosis who are homozygous for F508del aged 2 years and older following positive results from phase three trials. However, the improvement in CFTR function associated with lumacaftor/ivacaftor has only been studied in patients over 12 years of age, while the rescue potential in younger children is unknown.Methods: In a prospective study, we aimed to evaluate the effect of lumacaftor/ivacaftor on the CFTR biomarkers sweat chloride concentration and intestinal current measurement as well as clinical outcome parameters in F508del homozygous CF patients 2–11 years before and 8–16 weeks after treatment initiation.Results: A total of 13 children with CF homozygous for F508del aged 2–11 years were enrolled and 12 patients were analyzed. Lumacaftor/ivacaftor treatment reduced sweat chloride concentration by 26.8 mmol/L (p = 0.0006) and showed a mean improvement in CFTR activity, as assessed by intestinal current measurement in the rectal epithelium, of 30.5% compared to normal (p = 0.0015), exceeding previous findings of 17.7% of normal in CF patients homozygous for F508del aged 12 years and older.Conclusion: Lumacaftor/ivacaftor partially restores F508del CFTR function in children with CF who are homozygous for F508del, aged 2–11 years, to a level of CFTR activity seen in patients with CFTR variants with residual function. These results are consistent with the partial short-term improvement in clinical parameters

CFTR Cl− channel function in native human colon correlates with the genotype and phenotype in cystic fibrosis

BACKGROUND & AIMS: Cystic fibrosis (CF) is caused by over 1000 mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene and presents with a widely variable phenotype. Genotype-phenotype studies identified CFTR mutations that were associated with pancreatic sufficiency (PS). Residual Cl- channel function was shown for selected PS mutations in heterologous cells. However, the functional consequences of most CFTR mutations in native epithelia are not well established. METHODS: To elucidate the relationships between epithelial CFTR function, CFTR genotype, and patient phenotype, we measured cyclic adenosine monophosphate (cAMP)-mediated Cl- secretion in rectal biopsy specimens from 45 CF patients who had at least 1 non-DeltaF508 mutation carrying a wide spectrum of CFTR mutations. We compared CFTR genotypes and clinical manifestations of CF patients who expressed residual CFTR-mediated Cl- secretion with patients in whom Cl- secretion was absent. RESULTS: Residual anion secretion was detected in 40% of CF patients, and was associated with later disease onset (P < 0.0001), higher frequency of PS (P < 0.0001), and less severe lung disease (P < 0.05). Clinical outcomes correlated with the magnitude of residual CFTR activity, which was in the range of approximately 12%-54% of controls. CONCLUSIONS: Specific CFTR mutations confer residual CFTR function to rectal epithelia, which is related closely to a mild disease phenotype. Quantification of rectal CFTR-mediated Cl- secretion may be a sensitive test to predict the prognosis of CF disease and identify CF patients who would benefit from therapeutic strategies that would increase residual CFTR activity

The K+ Channel Opener 1-EBIO Potentiates Residual Function of Mutant CFTR in Rectal Biopsies from Cystic Fibrosis Patients

BACKGROUND: The identification of strategies to improve mutant CFTR function remains a key priority in the development of new treatments for cystic fibrosis (CF). Previous studies demonstrated that the K⁺ channel opener 1-ethyl-2-benzimidazolone (1-EBIO) potentiates CFTR-mediated Cl⁻ secretion in cultured cells and mouse colon. However, the effects of 1-EBIO on wild-type and mutant CFTR function in native human colonic tissues remain unknown. METHODS: We studied the effects of 1-EBIO on CFTR-mediated Cl⁻ secretion in rectal biopsies from 47 CF patients carrying a wide spectrum of CFTR mutations and 57 age-matched controls. Rectal tissues were mounted in perfused micro-Ussing chambers and the effects of 1-EBIO were compared in control tissues, CF tissues expressing residual CFTR function and CF tissues with no detectable Cl⁻ secretion. RESULTS: Studies in control tissues demonstrate that 1-EBIO activated CFTR-mediated Cl⁻ secretion in the absence of cAMP-mediated stimulation and potentiated cAMP-induced Cl⁻ secretion by 39.2±6.7% (P<0.001) via activation of basolateral Ca²⁺-activated and clotrimazole-sensitive KCNN4 K⁺ channels. In CF specimens, 1-EBIO potentiated cAMP-induced Cl⁻ secretion in tissues with residual CFTR function by 44.4±11.5% (P<0.001), but had no effect on tissues lacking CFTR-mediated Cl⁻ conductance. CONCLUSIONS: We conclude that 1-EBIO potentiates Cl⁻secretion in native CF tissues expressing CFTR mutants with residual Cl⁻ channel function by activation of basolateral KCNN4 K⁺ channels that increase the driving force for luminal Cl⁻ exit. This mechanism may augment effects of CFTR correctors and potentiators that increase the number and/or activity of mutant CFTR channels at the cell surface and suggests KCNN4 as a therapeutic target for CF

Hypoxic Epithelial Necrosis Triggers Neutrophilic Inflammation via IL-1 Receptor Signaling in Cystic Fibrosis Lung Disease

Rationale: In many organs, hypoxic cell death triggers sterile neutrophilic inflammation via IL-1R signaling. Although hypoxia is common in airways from patients with cystic fibrosis (CF), its role in neutrophilic inflammation remains unknown. We recently demonstrated that hypoxic epithelial necrosis caused by airway mucus obstruction precedes neutrophilic inflammation in Scnn1b-transgenic (Scnn1b-Tg) mice with CF-like lung disease

CFTR Regulates Early Pathogenesis of Chronic Obstructive Lung Disease in βENaC-Overexpressing Mice

<div><h3>Background</h3><p>Factors determining the onset and severity of chronic obstructive pulmonary disease remain poorly understood. Previous studies demonstrated that airway surface dehydration in βENaC-overexpressing (βENaC-Tg) mice on a mixed genetic background caused either neonatal mortality or chronic obstructive lung disease suggesting that the onset of lung disease was modulated by the genetic background.</p> <h3>Methods</h3><p>To test this hypothesis, we backcrossed βENaC-Tg mice onto two inbred strains (C57BL/6 and BALB/c) and studied effects of the genetic background on neonatal mortality, airway ion transport and airway morphology. Further, we crossed βENaC-Tg mice with CFTR-deficient mice to validate the role of CFTR in early lung disease.</p> <h3>Results</h3><p>We demonstrate that the C57BL/6 background conferred increased CFTR-mediated Cl⁻ secretion, which was associated with decreased mucus plugging and mortality in neonatal βENaC-Tg C57BL/6 compared to βENaC-Tg BALB/c mice. Conversely, genetic deletion of CFTR increased early mucus obstruction and mortality in βENaC-Tg mice.</p> <h3>Conclusions</h3><p>We conclude that a decrease or absence of CFTR function in airway epithelia aggravates the severity of early airway mucus obstruction and related mortality in βENaC-Tg mice. These results suggest that genetic or environmental factors that reduce CFTR activity may contribute to the onset and severity of chronic obstructive pulmonary disease and that CFTR may serve as a novel therapeutic target.</p> </div

Genetic background modifies early airway mucus obstruction and epithelial necrosis in neonatal βENaC-Tg mice.

<p>A) Longitudinal sections of tracheae from neonatal (3-day-old) wild-type (WT) and βENaC-Tg mice on the C57BL/6 and BALB/c background. Sections were stained with Alcian blue–periodic acid Schiff (AB-PAS) to determine the presence of mucus and goblet cells. Scale bars  = 200 µm. B–D) Summary of mucus content, as determined from measuring volume density of AB-PAS-positive material in the tracheal lumen (B), goblet cell numbers (C) and Transcript levels of Muc5b (D) (<i>n</i> = 4–13 mice per group). E) Airway histology from neonatal (3-day-old) WT and βENaC-Tg mice on the C57BL/6 and BALB/c background. Sections were stained with hematoxylin and eosin (H&E) and evaluated for degenerative airway epithelial cells (arrows). Scale bars  = 40 µm (upper panels) and 20 µm (lower panels). F) Summary of airway epithelial necrosis as determined from the number of degenerative epithelial cells per mm of the basement membrane (<i>n</i> = 9–11 mice per group). *<i>P</i><0.001 compared with WT mice on same strain background, ^†<i>P</i><0.05 compared with mice of same genotype on C57BL/6 background.</p

Survival of βENaC-Tg mice is modified by genetic background.

<p>Survival curves of βENaC-Tg mice and wild-type (WT) littermates on mixed (C3H/He x C57BL/6), C57BL/6 and BALB/c backgrounds (n = 36–59 mice per group). *<i>P</i><0.05 and ^†<i>P</i><0.01 (Kaplan-Meier survival analysis).</p

Genetic background modulates CFTR-mediated Cl⁻ secretion in airways of wild-type and βENaC-Tg mice.

<p>A–C) Effects of genetic background on transepithelial Cl- secretion were determined by adding bumetanide or CFTR_inh-172 to amiloride-pretreated tracheal tissues from neonatal wild-type (WT) and βENaC-Tg mice on the C57BL/6 and BALB/c background. A,B) Summary of bumetanide-sensitive I_sc (A) and CFTR_inh-172-sensitive I_sc (B) in the presence of amiloride (<i>n</i> = 12–23 mice per group). C) Summary of CFTR_inh-172-sensitive I_sc in the presence of amiloride and cAMP-dependent activation (IBMX/forskolin) (<i>n</i> = 5–11 mice per group). Data are presented as mean ± SEM. ^†<i>P</i><0.05 and ^††<i>P</i><0.01 compared with mice of same genotype on C57BL/6 background.</p

Genetic background modulates airway ion transport in wild-type and βENaC-Tg mice.

<p>A) Representative original recordings of the effects of amiloride and cAMP-dependent activation (IBMX/forskolin) on transepithelial voltage (V_te) and transepithelial resistance (R_te) across freshly excised tracheal tissues from neonatal (3-day-old) wild-type (WT) and βENaC-Tg mice on the C57BL/6 and BALB/c background. R_te was determined from V_te deflections obtained by pulsed current injection. (B–F) Summary of basal equivalent short-circuit current (I_sc) (B), amiloride-sensitive I_sc (C), amiloride-insensitive I_sc (D), cAMP-induced I_sc (E) and UTP-induced I_sc (F) in freshly excised tracheal tissues from neonatal WT and βENaC-Tg mice on the C57BL/6 and BALB/c background. Data are presented as mean ± SEM (<i>n</i> = 8–20 mice per group). *<i>P</i><0.05 and **<i>P</i><0.001 compared with WT mice on same strain background, ^†<i>P</i><0.05 and ^††<i>P</i><0.01 compared with mice of same genotype on C57BL/6 background.</p